The invention provides a solvent-free plasma method for depositing an adherent catechol and/or quinone functionalised layer to an inorganic or organic substrate from a precursor which comprises at least a ... [more ▼]

The invention provides a solvent-free plasma method for depositing an adherent catechol and/or quinone functionalised layer to an inorganic or organic substrate from a precursor which comprises at least a quinone group; a protected or unprotected catechol group; a molecule substituted by a quinone group and/or a protected or unprotected catechol group; and/or a natural or synthetic derivative of a catechol group and/or a quinone group; wherein the quinone group is a 1,2- benzoquinone group and the catechol group is a 1,2-dihydroxybenzene group. [less ▲]

Designing de novo proteins of more than 100 amino acids is still challenging. The creation of artificial (β/α)8-barrel proteins had only one successful example in literature, thank to use of internal ... [more ▼]

Designing de novo proteins of more than 100 amino acids is still challenging. The creation of artificial (β/α)8-barrel proteins had only one successful example in literature, thank to use of internal spatial symmetry. Here we present a protocol to design de novo (β/α)8-barrel proteins without symmetry restriction. First, the backbone was created in 4 steps: (I) Rosetta ParametricDesign produced an highly symmetric polyalanine scaffold with no loops; (II) Rosetta Fixed-Backbone Design used the previous output to substitute the alanines in all the position; (III) Loops were constructed with Modeller joining the terminus of the secondary structure elements and (IV) RosettaRelax performed relaxation, creating around 4000 different models. 28 backbone models were selected for the next steps of sequence design. To design the final proteins for experimental validation, 10 cycles of Rosetta Design and Relax were performed. In the first cycle only apolar amino acids were allowed in hydrophobic regions; in the next 6 cycles, amino acids were allowed based on the definition of 3 regions: core, boundaries and surface. All the amino acids were allowed in each position in the last 3 cycles. More than 10000 different sequences were created and analyzed in term of amino acid composition, sequence similarity with natural protein, secondary structure prediction, and molecular dynamics simulations. The 30 best candidate sequences have been selected for experimental verification. [less ▲]

The design of protein de novo is an emerging field in biochemistry, where artificial proteins are first designed in silico and then validated experimentally. This research, which rests mainly on our ... [more ▼]

The design of protein de novo is an emerging field in biochemistry, where artificial proteins are first designed in silico and then validated experimentally. This research, which rests mainly on our current understanding of protein structure, function, folding, stability and solubility, contributes to expand our knowledge of proteins in general. Our group has a long tradition in the design of artificial (β/α)8 -barrel proteins (called Octarellins). This fold is extremely interesting because it is widespread in nature (10% of the known proteins contain this fold) and in catalysis (it is present in 5/6 classes of enzyme). Here we present a protocol to design de novo (β/α)8-barrels with the more recent and best performing tools: Rosetta and Modeller (modelling softwares), and GROMACS (molecular dynamic simulations). First, 4000 artificial backbone structures were created with the use of modelling packages Rosetta and Modeller. 54 out of them were selected as targets for the following steps of sequence design and energy minimization (10 cycles), in order to find the best sequence to fit each target. More than 10000 different artificial sequences were created. Selection steps were performed in order to reduce the number of candidates for each target and the best ones were subjected to molecular dynamic simulation. Among this, 5 models were finally chosen for gene synthesis and experimental validation, and are currently being tested for expression in E. coli and preliminary purification. [less ▲]

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004[1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016 ... [more ▼]

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004[1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016/S0022-2836(02)01206-8[2]). The protein has been characterized by circular dichroism and fluorescence techniques, in order to obtain data related to its thermo and chemical stability. Moreover, the data for the secondary structure content studied by circular dichroism and infra red techniques is reported for the Octarellin V and V.1. Two crystallization helpers, nanobodies (http://dx.doi.org/10.1038/nprot.2014.039[3]) and alphaRep (http://dx.doi.org/10.1016/j.jmb.2010.09.048[4]), have been used to create stable complexes. Here we present the data obtained of the binding characterization of the Octarellin V.1 with the crystallization helpers by isothermal titration calorimetry. [less ▲]

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the ... [more ▼]

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (alphabetaalpha) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features. [less ▲]

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting ... [more ▼]

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with alpha-helical and beta-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70 degrees C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial alpha/beta protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility. [less ▲]

In this work, long-term antibacterial, antiadhesion, and antibiofilm activities are afforded to industrial stainless steel surfaces following a green and bio-inspired strategy. Starting from catechol ... [more ▼]

In this work, long-term antibacterial, antiadhesion, and antibiofilm activities are afforded to industrial stainless steel surfaces following a green and bio-inspired strategy. Starting from catechol bearing synthetic polymers, the film cross-linking and the grafting of active (bio)molecules are possible under environmentally friendly conditions (in aqueous media and at room temperature). A bio-inspired polyelectrolyte, a polycation-bearing catechol, is used as the film-anchoring polymer while a poly(methacrylamide)-bearing quinone groups serves as the cross-linking agent in combination with a polymer bearing primary amine groups. The amine/quinone reaction is exploited to prepare stable solutions of nanogels in water at room temperature that can be easily deposited to stainless steel. This coating provides quinonefunctionalized surfaces that are then used to covalently anchor active (bio) molecules (antibiofi lm enzyme and antiadhesion polymer) through thiol/ quinone reactions. [less ▲]

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in ... [more ▼]

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in medicine and industry, it would give us the ability to precisely engineer proteins to perform a specific function under a wider range of conditions. Despite impressive successes in the de novo protein design, designing a folded protein of more than 100 amino acids remains a challenge. In our lab, four generations of Octarellins, de novo polypeptides of more than two hundred amino acids modelled on the (beta/alpha)8 barrel fold, have been built and structurally characterized using biophysical and spectroscopic methods. The last generation of Octarellins was designed following a hierarchical method combining the specificity of rational design and the power of computational design. The resulting artificial protein, named Octarellin VI, was expressed in E. coli and purified from inclusion bodies. The biophysical characterization showed a monomeric protein, with a secondary structure level similar to the computationally designed model and thermostability. However, the poor solubility in bacteria and low stability of the protein at long term make impossible determine its structure to criticize the model. To improve these negative features, we performed a directed evolution process over the Octarellin, following the improvement at solubility level in the bacteria, thanks to the fusion of Octarellin to the fluorescent folding reporter GFP. After 8 cycles of directed evolution by Error Prone PCR technique, we obtained a most soluble protein, with a 92% of sequence identity with the original protein. This soluble variant is under study to characterize its structural features. The combination between in silico design and directed evolution process emerges as a powerful tool for protein engineering, showing be complementaries techniques and the information obtained by the whole process of design and posterior comparison between 3D structure of Octarellin with the computational model will allow to improve the algorithms for protein design. [less ▲]

A bio-inspired durable anti-biofilm coating was developed for industrial stainless steel (SS) surfaces. Two polymers inspired from the adhesive and cross-linking properties of mussels were designed and ... [more ▼]

A bio-inspired durable anti-biofilm coating was developed for industrial stainless steel (SS) surfaces. Two polymers inspired from the adhesive and cross-linking properties of mussels were designed and assembled from aqueous solutions onto SS surfaces to afford durable coatings. Trypsin, a commercially available broad spectrum serine protease, was grafted as the final active layer of the coating. Its proteolytic activity after long immersion periods was demonstrated against several substrata, viz. a synthetic molecule, N-a-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA), a protein, FTC-casein, and Gram-positive biofilm forming bacterium Staphylococcus epidermidis. [less ▲]

In this study, we report on the original synthesis and characterization of novel antimicrobial coatings for stainless steel by alternating the deposition of aqueous solutions of positively charged ... [more ▼]

In this study, we report on the original synthesis and characterization of novel antimicrobial coatings for stainless steel by alternating the deposition of aqueous solutions of positively charged polyelectrolytes micelles doped with silver based nanoparticles with a polyanion. The micelles are formed by electrostatic interaction between two oppositely charged polymers, a polycation bearing 3,4-dihydroxyphenylalanine units (DOPA, a major component of natural adhesives) and a polyanion (poly(styrene sulfonate), PSS) without using any block copolymer. DOPA units are exploited for their well-known ability to anchor to stainless steel and to form and stabilize biocidal silver nanoparticles (Ag0). The chlorine counter-anion of the polycation forms and stabilizes biocidal silver chloride nanoparticles (AgCl). We demonstrate that two layers of micelles (alternated by PSS) doped by silver particles are enough to impart to the surface a strong antibacterial activity against Gram-negative E. coli. Moreover, micelles that are reservoirs of biocidal Ag+ can be easily reactivated after depletion. This novel water-based approach is convenient, simple and attractive for industrial applications. [less ▲]

The goal of this paper was to establish the durability profile of antibacterial multilayer thin films under storage and usage conditions. Thin films were built on stainless steel (SS) by means of a layer ... [more ▼]

The goal of this paper was to establish the durability profile of antibacterial multilayer thin films under storage and usage conditions. Thin films were built on stainless steel (SS) by means of a layer-by-layer process alternating a negatively charged polyelectrolyte, polyacrylic acid, with a cationic antibacterial peptide, nisin. SS coupons coated with the antibacterial film were challenged under environmental and usage conditions likely to be encountered in real-world applications. The change in antibacterial activity elicited by the challenge was used as an indicator of multilayer film resistance. Antibacterial SS samples could be stored for several weeks at 4°C in ambient air and antibacterial films were resistant to dipping and mild wiping in water and neutral detergent. The multilayer coating showed some weaknesses, however, that need to be addressed. [less ▲]

A facile and green approach is developed to impart remarkable protection against corrosion to galvanized steel. A protecting multilayer film is formed by alternating the deposition of a polycation bearing ... [more ▼]

A facile and green approach is developed to impart remarkable protection against corrosion to galvanized steel. A protecting multilayer film is formed by alternating the deposition of a polycation bearing catechol groups, used as corrosion inhibitors, with clay that induces barrier properties. This coating does not affect the esthetical aspect of the surface and does not release any toxic molecules in the environment. [less ▲]

The synthesis of a poly(methacrylamide) bearing 3,4-dihydroxyphenylalanine inspired from the chemical composition of mussel adhesives will be presented. This homopolymer is designed to insure a multilayer ... [more ▼]

The synthesis of a poly(methacrylamide) bearing 3,4-dihydroxyphenylalanine inspired from the chemical composition of mussel adhesives will be presented. This homopolymer is designed to insure a multilayer film growth by covalent coupling during the layer-by-layer building with a homopolymer containing amino groups. The film cross-linking is initiated by adequately controlling both the redox state of the polymer and the pH of the solutions. This cross-linking is evidenced by solid-state 13C NMR with the occurrence of a typical signal of imine, traducing the Schiff base formation during film growth. The same coupling reaction is implemented for grafting an antibacterial peptide, Nisin, on a stainless steel substrate. Antibacterial activity against Bacillus subtilis is preserved even after long immersion time, as the result of the peptide covalent anchoring on the surface. All the processing steps, including the synthesis of the polymers and the peptide grafting, are performed in aqueous solutions under mild conditions. [less ▲]

The present paper relates a method for the separation of an insoluble inorganic powder out of a mixture of several insoluble powders with different chemical compositions, using genetically engineered ... [more ▼]

The present paper relates a method for the separation of an insoluble inorganic powder out of a mixture of several insoluble powders with different chemical compositions, using genetically engineered inorganic binding peptides (GEPI). GEPI are small peptides that recognize and specifically bind an inorganic solid material. This GEPI is anchored to magnetic beads for easy recovery of the powder of interest from the mixture. [less ▲]